"Glucose toxicity" accounts for insulin resistance in patients with uncontrolled type 1 diabetes and contributes to it in type 2 diabetes. It contributes to the vascular complications, the major causes of morbidity and mortality in diabetic patients. Sustained exposure of cells to high glucose increases flux via the hexosamine synthesis pathway, enhancing the production of UDP-N-acetyl glucosamine (UDP-GlcNAc), the substrate of O-GlcNAc transferase (OGT). OGT catalyzes the reversible, single addition of O-GlcNAc to specific Ser/Thr residues. O-GlcNAcylation and O-phosphorylation are often reciprocal. We have recently identified, for the first time, four sites of O-GlcNAcylation on IRS-1 (as well as 11 novel Ser/Thr phosphorylation sites) by mass spectrometry. Preliminary data suggest that O-GlcNAc may affect IRS-1 signal transduction. Our major objective in Spec. Aim 1 is to firmly establish whether the O-GlcNAc modification alters IRS-1 signaling. In in vitro studies Ser will be mutated to Ala at the four O-GlcNAc sites, singly and in combination, wild type and mutated IRS-1 will be expressed in HepG2 cells and in intact liver via adenovirus, and their interactions with IRS-1 binding partners in response to insulin or to IGF-1 studied. In in vivo studies endogenous mouse IRS-1 will be knocked out with shRNA adenovirus and substituted with wild type or mutant IRS-1-expressing adenovirus. The effect of these manipulations on glucose and insulin tolerance tests and the expression of hepatic gluconeogenic enzymes will be studied. In Spec Aim 2 studies will be continued in a model of high glucose/ low dose insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes. Insulin signaling to PI(3)K is largely maintained, but Akt activation is markedly impaired. Recent data indicate that PTEN protein expression is increased and insulin stimulated PtdIns(3,4,5)P3 is diminished. Stimulation of PTEN expression is inhibited by rapamycin. mTORC-1 activation clearly plays a role, cPKC may contribute, but JNK does not. The mechanism of enhanced PTEN expression, mTORC-1 action and the role of cPKC will be investigated. Several mechanisms which may synergize with or be mediated by mTOR will be addressed, including dysregulation of actin dynamics and possible activation of a phosphoprotein phosphatase(s). The analysis of the modus operandi in this model will be contrasted with the insulin resistance of glucose transport elicited by exposing cells to FFA. Insights gained from this model will be applied to L-6 myotubes and to intact rats made insulin resistant by chronic hyperglycemia. Understanding how different excess nutrients modify insulin's signaling may lead to the rational development of novel therapeutic targets.